a) Field of the Invention
This is a non-provisional application of U.S. provisional applications No. 60/185,063 filed on Feb. 25, 2000 and No. 60/225,062 filed on Aug, 17, 2000.
The entire content of a Sequence Listing titled “Sequence_Listing.txt,” created on Apr. 23, 2012 and having a size of 9 kilobytes, which has been submitted in electronic form in connection with the present application, is hereby incorporated by reference herein in its entirety.
The present invention relates to markers of endometriosis and more particularly to methods for determining likelihood of endometriosis in female subjects, to methods for grading endometriosis in females suffering from endometriosis and to methods for treating this disease. The invention is also concerned with polynucleotides, probes, primers and kits useful for reducing into practice the above-mentioned methods.
b) Brief Description of the Prior Art
Endometriosis is one of the most common gynecological disorders, affecting up to 10-15% of women of reproductive age. It is mainly associated with severe pelvic pain and/or infertility, but also with dysmenorrhea, dyspareunia, and several other symptoms such as intraperitoneal bleeding, back pain, constipation and/or diarrhea. Endometriosis is characterized by the implantation and growth of endometrial cells (which normally constitute the lining of the uterus) in extra-uterine sites, most frequently in the peritoneal cavity. The severity of the disease can be graded. According the American Society of Reproductive Medicine (ASRM), the disease is classified in four stages, namely, minimal (stage I), mild (stage II), moderate (stage III), and severe (stage IV). Although the etiology and pathogenesis of endometriosis remain unclear, the theory of retrograde menstruation is the most widely accepted to explain the presence of endometrial cells in ectopic sites. However, retrograde menstruation occurs in most women. Thus, a certain genetic potential or predisposition, present in the endometrial cells, might be responsible for the presence of the disease. Initially, this genetic potential may relate to mutations in the genome, but in addition, it may also lead to subsequent altered gene expression.
At present, direct visualization of the endometriotic lesions under surgical procedures (laparoscopy or laparotomy) is the only reliable method to diagnose endometriosis. However, this method is highly invasive (i.e. surgery under general anesthesia) and costly. The period of time between the onset of symptoms and disease diagnosis can be as long as 8 to 12 years. Ideally, the prospect to diagnose endometriosis more easily, rapidly, and as early as possible during the course of the disease would definitely reduce the number of years during which patients endure pain, infertility or other symptoms.
Based on this perspective, several investigators have sought to identify biological markers (proteinic and genetic) that could efficiently be used as predictive tools for endometriosis. However, to date, no one has been able to do so.
For instance, several proteins have also been shown to be differentially expressed in endometriosis. These include the tissue inhibitor of metaloproteinase-1 (TIMP-1), αvβ3 integrin, MCP-1, aromatase P450 and plasminogen activator-receptor and inhibitors. Unfortunately, the clinical relevance of these markers is uncertain since diagnostic parameters such as sensitivity and specificity of these candidate markers are still poorly defined.
Bcl-2 has been reported to be upregulated during the proliferative phase of the ovarian cycle in the eutopic endometrium of diseased women (Meresman et al. (2000) Fertil. Steril. 74(4): 760-6) as well as in endometriotic lesions in both phases of the cycle (Jones et al. (1998) Hum. Reprod. 13(12): 3496-502) and in macrophages from the peritoneal fluid of women having endometriosis (McLaren et al. (1997) Hum. Reprod. 12(1): 146-52). However, these results differ from our data presented herein in which a downregulation of Bcl-2 is observed in the eutopic tissue of women with endometriosis compared to disease-free women, independent of the phase of the ovarian cycle.
Connexin 43 (Cx43), a protein involved in gap junctions, has been reported to be aberrantly expressed in the glandular uterine epithelium of ectopic endometrial tissue in women with endometriosis (Regidor et al (1997) Mol. Hum. Reprod. 3:375-381). The goal of this study was solely to determine the hormonal regulation of connexins in endometriotic tissues, and consequently, this report did not analyze eutopic tissue in either women with endometriosis nor in disease-free women. Thus, findings in this study have little clinical or diagnostic relevance.
Human cyclooxygenase-2 (COX-2) is involved in prostaglandin synthesis, and, as a result, has been implicated in the growth and differentiation of endometrial stromal cells as well as during the implantation period necessary to establish pregnancy (Marions and Danielsson (1999) Mol. Hum. Reprod. 5:961-5). Due to its role in implantation during pregnancy, it has been postulated that COX-2 may be involved in the implantation of endometrial cells in ectopic sites, giving rise to endometriosis. However, to date, there have not been any conclusive reports demonstrating the role of COX-2 in endometriosis, nor that an alteration of its expression leads to the disease.
An increase in the expression at the protein level of heat shock protein 70 (HSP70) has been described in endometrial glandular cells of women having endometriosis and adenomyosis compared to a control group (Ota et al. (1997) Fertil Steril 68: 23-28). This result was obtained by immunohistochemistry. The same authors using the same technique showed that endothelial nitric oxide synthase (eNOS) and superoxide dismutase (SOD) were also up-regulated in the endometrium of patients with endometriosis or adenomyosis (Ota et al. (1998) Fertil Steril 69: 303-308; Ota et al. (1999) Fertil Steril 72: 129-134). However, these studies have limited clinical value because some of the experiments were not always carried out with an accurate technical approach, and because the markers were tested on a small number of patients yielding no statistically significant results. Furthermore, in Ota's studies, altered gene expression was found to occur in the ectopic tissue, as opposed to in the eutopic endometrium, and the results presented are therefore not industrially applicable.
Others groups studying gene expression have reported that the gluthatione S-transferase (GST) gene had a higher degree of polymorphism in endometriosis compared to a control group, and therefore represented an overall less-performing detoxification system which predisposed women to the disease (Baranova et al., (1999) Mol. Hum. Reprod. 5:636-641). These results reflect a genetic predisposition to have the disease rather than the likelihood of endometriosis.
Overall, no one has ever described any, methods for determining the likelihood of endometriosis in females, any methods for efficiently identifying females suffering from endometriosis, nor any methods for grading endometriosis in females suffering from the disease.
There is therefore a need for an alternative approach to laparoscopy or laparotomy to diagnose and determine the stage of endometriosis. More particularly, it would be highly desirable to be provided with methods wherein endometrial cells samples are assayed for expression levels of specific endometriosis-related genes (RNA or cDNA transcripts or their corresponding proteins), that are known to be differentially expressed in the endometrial cells of females with endometriosis (Endo group) compared to endometriosis-free females (Control group).
The present invention fulfils these needs and also other needs which will be apparent to those skilled in the art upon reading the following specification.